Bone Marrow-Derived Mesenchymal Stem Cells Expressing Thioredoxin 1 Attenuate Bleomycin-Induced Skin Fibrosis and Oxidative Stress in Scleroderma  Miao Jiang, Yiwu Yu, Jingying Luo, Qingyun Gao, Lili Zhang, Qiangxiong Wang, Jingjun Zhao  Journal of Investigative Dermatology  Volume 137, Issue 6, Pages 1223-1233 (June 2017) DOI: 10.1016/j.jid.2017.01.011 Copyright © 2017 The Authors Terms and Conditions

Figure 1 In vitro differentiation and typical phenotypes of BMSCs. (a) Surface antigen expression in mouse BMSCs at the third passage. Cells stained positive for mesenchymal stem cell markers CD29, CD44, CD54, CD90, and integrin β1. The endothelial markers CD31, CD34, and vWF were used as negative markers. ISO was used as a control (magnification = ×200). Scale bar = 20 μm. (b) Phase-contrast images showing BMSC morphology (magnification = ×200). Differentiation potential of BMSCs assessed by (c) Oil Red O and (d) alkaline phosphatase (magnification = ×200). scale bar = 20 μm. BMSC, bone marrow-derived mesenchymal stem cell; ISO, isotype control; vWF, Von Willebrand factor. Journal of Investigative Dermatology 2017 137, 1223-1233DOI: (10.1016/j.jid.2017.01.011) Copyright © 2017 The Authors Terms and Conditions

Figure 2 BMSCs transfected with the Trx-1 overexpression vector inhibit hypoxia-induced apoptosis. (a) Expression of Trx1 in BMSCs transfected with and without the recombinant Trx-1 plasmid detected by western blotting and real-time PCR. GAPDH expression was measured as an endogenous control. ***P < 0.001 versus control. (b) ROS in BMSCs cultured in normoxic or hypoxic conditions for 48 hours measured using DCF-DA. Immunofluorescence images show ROS (red fluorescence) and DAPI (blue fluorescence) staining. Magnification = ×200. Scale bar = 50 μm. (c) Average fluorescence intensity of DCF-DA in each group. N = 10. **P < 0.01, ***P < 0.001. NS = no significant differences. (d) Expression of oxidative stress-associated proteins NOX1 and NOX4 detected by western blotting. (e, f) Relative protein levels of NOX1 and NOX4. Values are represented as mean ± SEM (n = 3). **P < 0.01, ***P < 0.001. NS = no significant differences. (g) Flow cytometry analysis. Cells are double-labeled with annexin V-FITC and PI. (h) Relative apoptosis rate measured in five independent experiments. Relative protein levels are represented as mean ± SEM (n = 3). **P < 0.01, ***P < 0.001. NS = no significant differences. (i) Apoptosis-associated protein expression detected by western blotting. (j–l) Relative protein levels represented as mean ± SEM (n = 3). **P < 0.01, ***P < 0.001. NS = no significant differences. BMSC, bone marrow-derived mesenchymal stem cell; DCF-DA, 2′,7′-dichlorofluorescin diacetate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NOX, NADPH oxidase; PI, propidium iodide; ROS, reactive oxygen species; SCR, scrambled; SEM, standard error of the mean; Trx-1, thioredoxin 1. Journal of Investigative Dermatology 2017 137, 1223-1233DOI: (10.1016/j.jid.2017.01.011) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Trx-1-overexpressing BMSCs promote the formation of tubular-like structures in EPCs under hypoxic conditions. (a) Phase-contrast images showing the capacity of BMSCs to induce tube formation in EPCs. BMSCs were added at a 1:1 ratio with EPCs in serum-free DMEM under hypoxic conditions (magnification = ×200). Scale bar = 50 μm. (b) Tube length was quantified by densitometry. Values are represented as mean ± SEM (n = 10). $$$P < 0.001 versus EPC + BMSCs group. **P < 0.01, ***P < 0.001 versus EPC group. (c) Concentration of VEGF in the culture supernatant measured by ELISA. Values are represented as mean ± SEM (n = 5). $$$P < 0.001 versus EPC + BMSCs group. *P < 0.05, ***P < 0.001 versus EPC group. BMSC, bone marrow-derived mesenchymal stem cell; EPC, endothelial progenitor cell; SEM, standard error of the mean; Trx-1, thioredoxin 1; VEGF, vascular endothelial growth factor. Journal of Investigative Dermatology 2017 137, 1223-1233DOI: (10.1016/j.jid.2017.01.011) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Trx-1-overexpressing BMSCs inhibit fibrosis in myofibroblasts under hypoxic conditions. (a) Western blots showing expression of TGF-β, α-SMA, fibronectin, and collagen I. (b–e) Bar graphs representing quantitative differences in expression of TGF-β, α-SMA, fibronectin, and collagen I between groups. Values are represented as mean ± SEM (n = 3/group). ***P < 0.001 versus control group. $P < 0.05, $$$P < 0.001 versus BMSCs group. BMSC, bone marrow-derived mesenchymal stem cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SEM, standard error of the mean; α-SMA, α-smooth muscle actin; TGF-β, transforming growth factor-β; Trx-1, thioredoxin 1. Journal of Investigative Dermatology 2017 137, 1223-1233DOI: (10.1016/j.jid.2017.01.011) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Expression of Trx-1 promotes the survival of BMSCs and inhibits ROS-induced apoptosis. (a) Immunofluorescence showing survival of BMSCs overexpressing or not overexpressing Trx-1 at 3, 7, and 14 days after transplantation with GFP-labeled BMSCs (magnification = ×200). Scale bar = 50 μm. (b) Bar graphs showing the survival ratio of BMSCs in each group. Values are represented as mean ± SEM (n = 10). **P < 0.01, ***P < 0.001 versus BMSCs treatment group. (c) Oxidative stress in representative images from different treatment groups at 14 days after cell transplantation. Skin tissue sections are stained with DCF-DA (magnification = ×200). Scale bar = 50 μm. (d) Graph showing quantitative analysis (arbitrary units) of the average fluorescence intensity from DCF-DA fluorescence. Values are represented as mean ± SEM (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001 versus BMSCs treatment group. NS = no significant difference. (e–j) Oxidative stress-associated protein expression in (e–g) BMSCs and (h–j) Trx-1-overexpressing BMSCs measured by western blotting. Values are represented as mean ± SEM (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001 versus control. (k) Skin tissue sections were subjected to TUNEL staining 14 days after transplantation. (l) The ratio of TUNEL-positive (apoptotic) cells in each of the treatment groups is shown. Values are represented as mean ± SEM (n = 5). ***P < 0.001 versus normal (control) group. #P < 0.05, ###P < 0.001 versus PBS treatment. &P < 0.05 versus BMSCs treatment. Magnification = ×200. (m) Western blot showing Bcl-2, Bax, and caspase-3 expression. (n–p) Bar graphs showing quantitative differences in the expression of (n) Bcl-2, (o) Bax, and (p) caspase-3 between treatment groups. Values are represented as mean ± SEM (n = 5). *P < 0.05, ***P < 0.001 versus control group. ##P < 0.01, ###P < 0.001 versus PBS treatment group. $P < 0.05 versus BMSCs treatment group. BMSC, bone marrow-derived mesenchymal stem cell; DCF-DA, 2′,7′-dichlorofluorescin diacetate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; NOX, NADPH oxidase; PBS, phosphate buffered saline; ROS, reactive oxygen species; SEM, standard error of the mean; Trx-1, thioredoxin 1. Journal of Investigative Dermatology 2017 137, 1223-1233DOI: (10.1016/j.jid.2017.01.011) Copyright © 2017 The Authors Terms and Conditions

Figure 6 Origin of endothelial cells in skin tissue sections. GFP-labeled BMSCs were detected in skin tissue sections by GFP immunofluorescence. (a) Representative images of transplant sections stained with a Cy3-labeled mAb targeted against the endothelial cell marker CD31 with PE (red). Merged images showing colocalization of vWF and GFP, indicating differentiation of endothelial cells from GFP-labeled BMSCs (magnification = ×200). Scale bar = 50 μm. (b) Expression of VEGF in skin tissue samples measured by western blotting. (c) Bar graphs showing quantitative differences in expression of VEGF. Values are represented as mean ± SEM (n = 5). ***P < 0.001 versus control group. ##P < 0.01, ###P < 0.001 versus PBS treatment. $P < 0.05 versus BMSCs treatment group. BMSC, bone marrow-derived mesenchymal stem cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; PBS, phosphate buffered saline; PE, phycoerythrin; SEM, standard error of the mean; Trx-1, thioredoxin 1; VEGF, vascular endothelial growth factor; vWF, Von Willebrand factor. Journal of Investigative Dermatology 2017 137, 1223-1233DOI: (10.1016/j.jid.2017.01.011) Copyright © 2017 The Authors Terms and Conditions

Figure 7 Transplantation of Trx-1-overexpressing BMSCs prevents dermal thickening and fibrosis induced by bleomycin administration. (a) Representative images of HE-stained sections of male mouse skin tissue after treatment with BMSCs or Trx-1-overexpressing BMSCs for 2 weeks. Original magnification = ×100. Scale bar = 10 μm. (b) Images showing Masson’s trichrome staining and quantification of skin tissue samples from different treatment groups (magnification = ×200). Scale bar = 50 μm. (c) Representative images of TGF-β immunofluorescence analysis in skin tissue samples from mice in different treatment groups (magnification = ×400). Scale bar = 20 μm. (d) Western blot showing expression of TGF-β, α-SMA, fibronectin, and collagen I. The bar graphs represent quantitative differences in expression of TGF-β, α-SMA, fibronectin, and collagen I between treatment groups. Values are represented as mean ± SEM (n = 5 mice/group). **P < 0.01, ***P < 0.001 versus control group. #P < 0.05, ##P < 0.01, ###P < 0.001 versus PBS treatment group. $P < 0.05 versus BMSCs treatment group. BMSC, bone marrow-derived mesenchymal stem cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HE, hematoxylin and eosin; PBS, phosphate buffered saline; SEM, standard error of the mean; α-SMA, α-smooth muscle actin; TGF-β, transforming growth factor-β; Trx-1, thioredoxin 1. Journal of Investigative Dermatology 2017 137, 1223-1233DOI: (10.1016/j.jid.2017.01.011) Copyright © 2017 The Authors Terms and Conditions